中国蛤蜊神经系统谷氨酸递质及受体通路对无机汞暴露的响应研究

Glutamate is one of the most important excitatory amino acid neurotransmitters in the nervous system, which activates two main types of postsynaptic receptors (ionotropic glutamate receptors and metabotropic glutamate receptors). The glutamate receptor-regulated network was found to play a key role in the organisms to environmental stressors. In previous studies, we have found that the levels of glutamate in Ruditapes philippinarum was significantly elevated in HgCl2-exposed groups than control using proton nuclear magnetic resonance (NMR) spectroscopy-based metabolomics, which suggested that mercuric chloride perhaps cause glutamate-induced neurotoxicity in shellfish. Due to its advanced nervous systems in marine bivalve molluscs, Mactra chinensis Philippi is a preferable bioindicator used to explore the neurotoxicity of HgCl2 in this project. In the present study, we aim to: (1) detect the alteration of neurotransmitter （glutamate） by high performance liquid chromatography; (2) investigate the protein changes of ionotropic and metabotropic glutamate receptors by Western blot; (3) analyse the gene expression of two types of glutamate receptors by real-time quantitative reverse transcription polymerase chain reaction; (4) compare the proteomic responses induced by HgCl2 in the nervous system of Mactra chinensis Philippi using iTRAQ-based proteomics. Based on these results, we attempt to characterize the specific glutamate receptor responded to HgCl2 exposure and elucidate the underlying molecular mechanisms in Mactra chinensis Philippi. The results of this study will not only contribute to the further knowledge of defense mechanisms in glutamatergic systems of mollusks, but also provide a reference for better understanding the degeneration of biological resources in a polluted marine environment.

谷氨酸是最主要的兴奋性氨基酸类神经递质，其介导的谷氨酸受体通路在生物体感知环境变化中发挥着重要作用。申请人前期研究已发现，无机汞（HgCl2）暴露导致贝类谷氨酸含量的特异性升高，可引起贝类体内谷氨酸介导的神经毒性。本项目以无机汞为暴露污染物，选取神经系统较发达的双壳贝类中国蛤蜊为研究对象，通过高效液相色谱、实时荧光定量PCR、免疫印迹等技术，分析汞对中国蛤蜊神经组织中谷氨酸含量的影响，以及谷氨酸受体蛋白在基因及蛋白水平的表达变化特征，解析谷氨酸能神经系统对汞暴露的生理响应；在此基础上，采用基于iTRAQ的蛋白质组学技术探究神经系统内参与调控的关键蛋白分子，构建神经系统的蛋白网络调控图谱，以期阐明贝类神经系统对无机汞响应的分子机制。研究结果可为深入探索污染物暴露下的海洋生物资源响应及其衰退机制提供一定的科学依据。

重金属对海洋生物的毒理效应及致毒机制已成为海洋生态环境领域的研究热点之一。本项目以无机汞为暴露污染物，以中国蛤蜊为研究对象，主要采用基于iTRAQ的差异蛋白质组学技术，解析中国蛤蜊神经系统对汞暴露的响应特征及分子机制。通过研究主要得到：（1）中国蛤蜊鳃、肝、足、闭壳肌、外套膜及神经组织对汞都有不同程度蓄积。（2）汞暴露后中国蛤蜊神经递质谷氨酸含量增加，乙酰胆碱和一氧化氮含量下降，一氧化氮合酶及钙调神经磷酸酶活性增强，并引起钙离子超载，汞引起中国蛤蜊神经递质的合成及释放障碍。（3）通过蛋白质组学实验，共筛选出947个差异蛋白，该组差异蛋白主要为胞质核糖体、细胞质、胞质部分、核糖体亚基等细胞组分的相关蛋白，主要参与包括肽代谢、翻译、肽生物合成、细胞酰胺代谢等生物过程，参与的分子功能包括核糖体的结构组成、结构分子活性、RNA结合、阴离子结合等。KEGG分析显示，差异蛋白主要为核糖体、内吞作用、丙酸新陈代谢等相关蛋白。（4）通过对代谢产物进行OPLS-DA分析，谷氨酸及谷氨酰胺代谢物发生显著性改变，与其他研究结果相辅相成，验证了谷氨酸能神经通路对汞的毒性响应。研究结果阐明了典型双壳贝类神经系统对无机汞响应的分子机理，对揭示贝类神经系统的环境适应机制具有一定的理论意义，同时为污染物的海洋环境风险评价提供一定的理论依据。